J. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

DIFFERENTIATION OF CLARIAS BATRACHUS, C. GARIEPINUS AND BY PCR-SEQUENCING OF MITOCHONDRIAL 16S rRNA GENE

MOHAMMAD SHAMIMUL ALAM1, HAWA JAHAN2, ROWSHAN ARA BEGUM2, REZA M. SHAHJAHAN2* 1Department of Botany Dhaka University Dhaka, Dhaka-1000 2Department of Zoology, University of Dhaka, Dhaka 1000, Bangladesh

Abstract

Heteropneustes fossilis, Clarias batrachus and C. gariepinus are three major of ecological and economic importance. Identification of these fish becomes a problem when the usual external morphological features of the fish are lost or removed, such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCR- sequencing provides accurate alternative means of identification of individuals at species level. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCR amplification and compared with the same gene sequences available from other geographical regions. Multiple sequence alignment of the 16S rRNA gene fragments of the species has revealed polymorphic sites which can be used to differentiate these three species from one another and will provide valuable insight in choosing appropriate restriction enzymes for PCR-RFLP based identification in future.

Key words: PCR-sequencing, 16S rRNA, Heteropneustes, Clarias, Species identification

Introduction Catfishes of the Heteropneustes and Clarias (Siluriformes: Heteropneustidae, Clariidae) are commercially and ecologically important groups of freshwater fishes. These closely related species share more or less conservative external morphology and are difficult to identify when usual external features are missing. For instance, during fish processing into portions of flesh, such as in canned fish, species identification could be impossible due to loss of identifying characteristics (Quinteiro et al. 1998). Also, newly hatched fish larvae are often difficult to differentiate based on morphology and alternative method is in use (Lindstrom 1999). In addition, species complexes are difficult to identify based on morphology alone. Molecular techniques, especially DNA-based species identification techniques are better alternatives to morphology-based ones (Teletchea 2009). Analysis of polymerase chain reaction (PCR) amplified DNA fragments can provide an accurate alternative means of identification of individuals to genes or species level (Hubalkova et al. 2008, Jans et al.

1Corresponding author: E-mail: [email protected] 52 Alam et al.

2012, Kochzius et al. 2010, Lakra et al. 2011, Scheidegger et al. 2009 and Teletchea 2009). Among many PCR-based approaches, PCR-sequencing is found to provide highest amount of information and has been widely used now-a-days (Hajibabaei et al. 2007, Rehbein 2013, Zeng et al. 2013 and Zhuang et al. 2013). PCR-RFLP (Restriction Fragment Length Polymorphism) is another good choice for differentiation of species with known sequence. PCR-RFLP of different genes was demonstrated to detect inter- and intra-specific variations in several (Di Finizio et al. 2007 and Duduk et al. 2013). PCR-RFLP analysis is faster and more cost effective. Mitochondrial DNA (mtDNA) is maternally inherited and does not go through recombination event. Single nucleotide polymorphism in the mitochondrial genome increase the power of discrimination among the individuals (Coble et al. 2004). mtDNA genetic markers, such as Cytochrome oxidase I (COI), 16S and 12S rRNA genes have been widely used as tools to distinguish within and among species (Di Finizio et al. 2007, Klossa et al. 2002 and Wolf et al. 2000). Recently, 16S rRNA gene sequences are reported to be particularly useful in identifying different species (Sarri et al. 2014). In this background, the main objectives of the present investigations are (1) to compare the 16S rRNA gene sequences of three catfishes from Bangladesh with the existing sequences of those in the GenBank database, (2) to compare sequences among the three selected catfishes and (3) to explore the possibility of identifying these three species using PCR-RFLP on 16S rRNA gene sequence.

Materials and Methods Sample Collection and DNA Extraction: H. fossilis (locally known as Shing), C. batrachus (Magur) and C. gariepinus (African Magur) were collected from local fish markets (Dhaka, Bangladesh). DNA was extracted from 50 mg muscle tissue of each fish species following a standard method (Doyle and Doyle 1990) with modifications. CTAB extraction buffer (1M 10 ml Tris-HCl [pH 8], 0.5M 4ml Sodium-EDTA [pH 8], 8.18 g NaCl, 2g CTAB, dH₂O) and 10 µl of 20 mg/mL proteinase K were added with minced meat sample. The mixture was incubated at 55°C for 2 h. After digestion, the samples were centrifuged at 13000 rpm for 5 min and equal volume of Phenol: Chloroform was added to the supernatant. Then, DNA was precipitated with ethanol and dissolved in distilled water. PCR amplification and Sequencing: Universal primers for PCR of the 16S rRNA gene, 16Sar-L (5’-CCGGTCTGAAAAAAACAT) and 16Sar-H (5’-CCGGTCTGAA CTCAG ATCACGT) have been utilized (Palumbi 1996). Standard PCR was performed using PCR Master Mix (Promega). Temperature regimes for PCR amplifications were as follows – initial denaturation at 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30s and 72°C on the last cycle. PCR products were run in an agarose gel containing ethidium bromide. The electrophoresis was performed at 90 V for 30 min and Differentiation of Clarias batrachus 53

DNA bands were visualized on a UV transilluminator and photographed. Then, PCR amplicons were purified using a purification kit (FavorGen) and sequenced. Bioinformatics: 16S rRNA gene sequences of experimental fish species and existing sequences of related fish species were collected from GenBank database (NCBI). Suitable portion of each sequence was taken for further observation. A multiple sequence alignment was performed using ClustalW software to observe the polymorphic sites present among the sequences. Restriction endo-nuclease cutting sites were searched in the sequences of the 16S rRNA fragment analyzed further. ‘Serial Cloner’ software was used to observe restriction sites.

Results and Discussion Comparison of 16S rRNA gene sequence of three catfishes from Bangladesh with the available sequences from GenBank database: Partial sequences of 16S rRNA genes (5 Clarias spp. and 2 Heteropneustes spp.) were retrieved from GenBank database (Table 1). Suitable portion of DNA sequence (560 bp) was analyzed further. A multiple sequence alignment was done by SeaView to compare the sequences (Gouy et al. 2010) (Fig. 1). After comparing these sequences in different individuals, 55 out of 560 nucleotide bases of the sequence were found polymorphic. Among these polymorphic sites, nine were selected as positions with diagnostic value at genus level (Table 2). In seven positions (137, 219, 325, 367, 439, 443, 463) genus Heteropneustes and genus Clarias show nucleotide variations. In another two positions (242, 328), Clarias has particular nucleotide bases though deletion was observed in Heteropneustes. These diagnostic positions were selected using the criteria that these positions did not show intra-specific variability. In some positions, inter-species variations were also observed. 16S rRNA gene sequence of H. fossilis differs from that of H. microps in positions 34, 181, 241, 261, 313, 326, 331, 336, 338, 351, 361. Besides, H. fossilis sequence from Bangladesh differs from that of other geographical regions in the positions 271 and 377. Mitochondrial DNA diversity due to geographical differences has been reported elsewhere (Linares et al. 2009).

Table 1. List of 16S rRNA sequences used in this study. Scientific name Common name GenBank Accession Collected Clarias batrachus Walking Catfish (Magur) KF997532.1*Numbers Bangladeshfrom C. batrachus Walking Catfish JQ699193 India C. gariepinus North African Catfish (African KJ819942* Bangladesh C. gariepinus NorthMagur) African Catfish JQ699188 India C. dussumieri - JQ699198.1 India C. fuscus White spotted Clarias JN020056.1 China C. gabonensis - JX899749.1 Gabon Heteropneustes Stinging Catfish (Shing) KJ819943* Bangladesh H.fossilis fossilis Stinging Catfish FN677932 India H. fossilis Stinging Catfish GQ411079 India H. microps Stinging Catfish FJ432686 India *Sequences submitted to GenBank from this study. C. dussumieri

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Sequence comparison among three selected catfishes: Fragments of mtDNA of three selected catfishes (Clarias batrachus, C. gariepinus and Heteropneustes fossilis) were sequenced. The GenBank accession numbers are KF997532.1, KJ819942 and KJ819943, respectively. A multiple sequence alignment was performed by SeaView (Gouy et al. 2010). Twenty nine out of 560 bases of the sequence were found polymorphic. Among the polymorphic sites, 15 have diagnostic value at genus level, i.e, genus Clarias and genus Heteropneustes can be differentiated by these 15 nucleotide variations (Table 3). Comparing the sequences, polymorphic sites were also observed between two species belonging to the genus Clarias. Fifteen positions were found which have diagnostic value at species level (Table 4). Two species of Clarias (C. batrachus and C. gariepinus) can be differentiated by this nucleotide variation.

Fig. 1. Multiple sequence alignment of 16S rRNA gene fragments of Clarias spp. and Heteropneustes spp. Some representative polymorphic sites are indicated by the nucleotide position numbers of Clarias batrachus (Cbat) sequence on the top. Differentiation of Clarias batrachus 55

Table 2. Polymorphic sites observed between two catfish genus from Bangladesh with the existing sequences of those in the GenBank database in 16S rRNA gene fragment with diagnostic value. Position Heteropneustes spp. Clarias spp. 137 T C 219 A C 242 nucleotide deleted T or A 325 A G 328 nucleotide deleted A or C 367 C T 439 C T 443 T C 463 G A

Table 3. Polymorphic sites observed among three native catfishes from Bangladesh in 16S rRNA gene fragment with diagnostic value (differentiation at genus level). Position Clarias spp. Heteropneustes spp. 137 C T 219 C A 271 C T 313 A G 325 G A 338 C T 359 A G 361 T C 367 A G 389 C T 393 C T 438 T C 442 C T 462 A G

Possible differentiation of three species using PCR-RFLP on 16S rRNA gene: Partial 16S rRNA sequences of the three catfishes were tested with various restriction enzymes using open source molecular biology software Serial Cloner (http://serialbasics.free.fr/ Serial_Cloner.html). Some of the enzymes were found which are able to differentiate the selected species. This bioinformatic analysis has identified three restriction enzymes that are useful for distinguishing among the catfishes (Table 5). Restriction enzymes for RFLP analysis were selected based on two criteria. Firstly, it has the ability to provide differentiation among the species and secondly, it produces fragments of suitable sizes easy to visualize in agarose gel. PCR-RFLP using these three restriction enzymes, AfaI, NspI and PasI can separate two genus Heteropneustes and Clarias, although there are some other enzyme options as well. Besides, two species of the same genus Clarias (C. batrachus and C. gariepinus) can also be differentiated by these enzymes. So, the restriction enzymes AfaI, NspI and PasI are candidates of highest diagnostic value for the selected three species. Once experimental proof is obtained, PCR-RFLP using these three enzymes will help for easy and cheaper differentiation of the selected catfishes.

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Table 4. Polymorphic sites observed between two Clarias species in 16S rRNA gene fragment with diagnostic value (differentiation at species level). Position C. batrachus C. gariepinus 181 C A 184 G A 240 T C 242 T A 247 A G 261 T C 327 C A 330 A deletion 336 A G 355 C T 377 T C 399 T C 418 C T 560 C T

Table 5. Restriction enzymes (RE) which allow differentiation among three species. RE with cutting sites Position C. batrachus C. gariepinus H. fossilis AfaI 184 G A A GTAC (digested) (not digested) (not digested) CATG NspI 389 C C T RCATGY (not digested) (not digested) (digested) YGTACR PasI 418 C T T CCCWGGG (digested) (not digested) (not digested) GGGWCCC Both mitochondrial and nuclear gene sequences are used for the purpose of molecular species identification (Rehbein 2013 and Hajibabaei et al. 2007). Mitochondrial genes, such as, Cytochrome oxidase I (COI), 16S and 12S rRNA genes are commonly used markers for molecular species identification (Cawthorn et al. 2012 Di Finizio et al. 2007 and Zhuang et al. 2013). Among the three mitochondrial genes, 16S rRNA gene has been more suitable because it is highly conserved among the vertebrates and has lower sequence variation among the populations of same species (Kitano et al. 2007, Di Finizio et al. 2007, Cawthorn et al. 2012 and Kochzius et al. 2010). Compared to COI, 16S rRNA gene has been reported to have lower K2P distances (about 10 fold) within same species (Cawthorn et al. 2012 and Lakra et al. 2011). In a study of eight pecoran species, 16S rRNA gene was found to have a larger number of species-specific polymorphic sites compared to the cytochrome b gene indicating the usefulness of 16S rRNA gene for species identification (Guha et al. 2006). In this circumstance, analyzing partial sequences of 16S rRNA gene for molecular differentiation of three selected catfishes of Bangladesh is a timely work, if not late. The results also prove that how this work can help to differentiate these three species. Moreover, using appropriate restriction enzymes Differentiation of Clarias batrachus 57

(as indicated by this study), these three species can be separated by RFLP as well, which will be a less time-consuming and cheaper practice.

Acknowledgements We acknowledge Center for Advanced Research in Sciences (CARS), University of Dhaka for providing sequencing services. Besides, we thank the Department of Zoology, University of Dhaka for supplying necessary chemicals and reagents of this research.

References Cawthorn, D. M., H. A. Steinman and R. C. Witthuhn. 2012. Evaluation of the 16S and 12S rRNA genes as universal markers for the identification of commercial fish species in South Africa. Gene. 491 (1):40-8. Coble, M. D., R. S. Just, J. E. O'Callaghan, I. H. Letmanyi, C. T. Peterson, J. A. Irwin and T. J. Parsons. 2004. Single nucleotide polymorphisms over the entire mtDNA genome that increase the power of forensic testing in Caucasians. Int J Legal Med. 118 (3):137-46. Di Finizio, A., G. Guerriero, G. L. Russo and G. Ciarcia. 2007. Identification of gadoid species (Pisces, Gadidae) by sequencing and PCR–RFLP analysis of mitochondrial 12S and 16S rRNA gene fragments. European Food Research and Technology. 225 (3-4):337-344. Doyle, J.J. and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus. 12:13–15. Duduk, B., S. Paltrinieri, I. M. Lee and A. Bertaccini. 2013. Nested PCR and RFLP analysis based on the 16S rRNA gene. Methods Mol Biol. 938:159-71. Gouy, M., S. Guindon and O. Gascuel. 2010. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol. 27 (2):221-4. Guha, S., S. P. Goyal and V. K. Kashyap. 2006. Genomic variation in the mitochondrially encoded cytochrome b (MT-CYB) and 16S rRNA (MT-RNR2) genes: characterization of eight endangered Pecoran species. Anim Genet. 37 (3):262-5. Hajibabaei, M., G. A. Singer, P. D. Hebert and D. A. Hickey. 2007. DNA barcoding: how it complements , molecular phylogenetics and population genetics. Trends Genet. 23 (4):167-72. Hubalkova, Z., P. Kralik, J. Kasalova and E. Rencova. 2008. Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA. J Agric Food Chem. 56 (10):3454-9. Jans, C., C. Lacroix and L. Meile. 2012. A novel multiplex PCR/RFLP assay for the identification of Streptococcus bovis/Streptococcus equinus complex members from dairy microbial communities based on the 16S rRNA gene. FEMS Microbiol Lett. 326 (2):144-50. Kitano, T., K. Umetsu, W. Tian and M. Osawa. 2007. Two universal primer sets for species identification among vertebrates. Int J Legal Med. 121 (5):423-7. Klossa, K.E., V. Papasotiropolos, G. Kilias and S. Alahiotis. 2002. Authentication of Messolongi (Greece) fish role using PCR-RFLP analysis of 16S rRNA mtDNA segment. Food Control. 13: 169-72. doi: 10.1016/S0956-7135(01)00097-4 Kochzius, M., C. Seidel, A. Antoniou, S. K. Botla, D. Campo, A. Cariani, E. G. Vazquez, J. Hauschild, C. Hervet, S. Hjorleifsdottir, G. Hreggvidsson, K. Kappel, M. Landi, A. Magoulas, V. Marteinsson, M. Nolte, S. Planes, F. Tinti, C. Turan, M. N. Venugopal, H. Weber and D. Blohm. 2010. Identifying Fishes through DNA Barcodes and Microarrays. PLoS ONE, 5 (9):e12620. Lakra, W. S., M. S. Verma, M. Goswami, K. K. Lal, V. Mohindra, P. Punia, A. Gopalakrishnan, K. V. Singh, R. D. Ward and P. Hebert. 2011. DNA barcoding Indian marine fishes. Mol Ecol Resour. 11 (1):60-71.

58 Alam et al.

Linares, M. C., I. D. Soto-Calderon, D. C. Lees and N. M. Anthony. 2009. High mitochondrial diversity in geographically widespread butterflies of Madagascar: a test of the DNA barcoding approach. Mol Phylogenet Evol. 50 (3):485-95. Lindstrom, D. P. 1999. Molecular Species Identification of Newly Hatched Hawaiian Amphidromous Gobioid Larvae. Mar Biotechnol (NY). 1 (2):167-174. Palumbi, S.R. 1996. Nucleic acids II: The polymerase chain reaction. In: Molecular Systematics. (ed. D.M. Hillis, C. Moritz and B.K. Mable): pp. 205-247, Sinauer, Sunderland, Massachusetts. Quinteiro, J., C. G. Sotelo, H. Rehbein, S. E. Pryde, I. Medina, R. I. Pérez-Martín, M. Rey-Méndez and I. M. Mackie. 1998. Use of mtDNA Direct Polymerase Chain Reaction (PCR) Sequencing and PCR−Restriction Fragment Length Polymorphism Methodologies in Species Identification of Canned Tuna. Journal of Agricultural and Food Chemistry. 46 (4):1662-1669. Rehbein, H. 2013. Differentiation of fish species by PCR-based DNA analysis of nuclear genes. European Food Research and Technology. 236 (6):979-990. Sarri, C., C. Stamatis, T. Sarafidou, I. Galara, V. Godosopoulos, M. Kolovos, C. Liakou, S. Tastsoglou and Z. Mamuris. 2014. A new set of 16S rRNA universal primers for identification of animal species. Food Control. 43 (0):35-41. Scheidegger, E. M., S. A. Fracalanzza, L. M. Teixeira and P. Cardarelli-Leite. 2009. RFLP analysis of a PCR-amplified fragment of the 16S rRNA gene as a tool to identify Enterococcus strains. Mem Inst Oswaldo Cruz. 104 (7):1003-8. Teletchea, F. 2009. Molecular identification methods of fish species: reassessment and possible applications. Reviews in Fish Biology and Fisheries. 19 (3):265-293. Wolf, C., M. Burgener, P. Hubner and J. Luthy. 2000. PCR-RFLP analysis of mitochondrial DNA: differentiation of fish species. Lebensmittel-Wissenschaft und technologie. 33: 144-50. Zeng, Y. H., M. Koblizek, Y. X. Li, Y. P. Liu, F. Y. Feng, J. D. Ji, J. C. Jian and Z. H. Wu. 2013. Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping. J Appl Microbiol. 114 (2):433-47. Zhuang, X., M. Qu, X. Zhang and S. Ding. 2013. A comprehensive description and evolutionary analysis of 22 grouper (perciformes, epinephelidae) mitochondrial genomes with emphasis on two novel genome organizations. PLoS ONE. 8 (8):e73561.

(Received revised manuscript on 28/2/2015)